Human growth hormone (hGH) sequences were also inserted into the .... thickening and hair follicle density; (ii) none of the KGF-expressing animals had fat ...
The EMBO Journal vol.12 no.3 pp.973-986, 1993
Targeting expression of keratinocyte growth factor to keratinocytes elicits striking changes in epithelial differentiation in transgenic mice Lifei Guo, Qian-Chun Yu and Elaine Fuchs1 Howard Hughes Medical Institute, Department of Molecular Genetics and Cell Biology, The University of Chicago, Chicago, IL 60637, USA 'Corresponding author Communicated by E.A.Nigg
Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. Synthesized by cells of the dermal component of skin, KGF's potent mitogenic activity is on the epidermal component, which harbors the receptors for this factor. To explore the possible role of KGF in mesenchymal -epithelial interactions in skin, we used a human keratin 14 promoter to target expression of human KGF cDNA to the stratified squamous epithelia of transgenic mice. Mice expressing KGF in their epidermis typically appeared frail and weak, and often had grossly wrinkled skin. These mice exhibited a gross increase in epidermal thickness accompanied by alterations in epidermal growth and differentiation. Most remarkably, animals displayed several striking and unexpected changes, including a marked suppression of hair follicle morphogenesis and suppression of adipogenesis. With age, some animals developed gross transformations in the tongue epithelium and in epidermis. In addition, they exhibited elevated salivation and their salivary glands showed signs of altered differentiation. Collectively, our findings provide new and important insights into the roles of KGF, implicating this potent growth factor in eliciting global effects not only on growth, but also on development and differentiation, of skin and other tissues. In particular, KGF seems to interfere with signalling of some mesenchymal-epithelial interactions. Key words: epidermis/hyperproliferation/keratinocyte growth factor/terminal differentiation
Introduction Keratinocyte growth factor (KGF) is made by stromal fibroblasts and belongs to the seven-member fibroblast growth factor (FGF) family (Finch et al., 1989; Aaronson et al., 1991). KGF is synthesized as a single peptidecontaining precursor that is cleaved upon secretion to liberate mature KGF (Finch et al., 1989). It differs from other FGFs in that its mitogenic activity appears to be specific for keratinocytes, and not fibroblasts or endothelial cells (Finch et al., 1989). This paracrine pathway for stimulating keratinocyte growth is activated by an alternative splicing variant of the surface receptor FGFR2, whose tyrosine kinase is inducible by either KGF or aFGF (Rubin et al., 1989; Bottaro et al., 1990; Miki et al., 1992; Peters et al., 1992). In vitro studies have shown that the stimulating effects of Oxford University Press
KGF on DNA synthesis in keratinocytes is 2- to 10-fold stronger than that of autocrine keratinocyte growth factors such as TGFa and EGF (Rubin et al., 1989). Together with the finding that the expression of KGF mRNA (and presumably protein) is dramatically elevated after skin injury (Werner et al., 1992), KGF appears to be a major factor in promoting epidermal cell proliferation. Little is known about the possible effects of KGF on other cell types, organs or tissues, although the FGFR2 receptor is expressed in epithelial tissues at times in embryonic development when epidermal-mesenchymal interactions may play a role in organogenesis (Orr-Urtreger et al., 1991; Peters et al., 1992). The skin is a complex organ, composed of many different cell types. The major structural components of the skin are the superficial epidermis, its appendages, the underlying dermis and the cutaneous fat (Fitzpatrick et al., 1987). To coordinate and maintain the appropriate proportions and differentiation states of cell types within the skin, there must be intricate and intertwined mechanisms of intercellular signalling. In this regard, it is well established that certain factors produced and secreted by mesenchymal cells are necessary for epidermal cells to grow, develop and differentiate (Wessels, 1977; Dhouailly et al., 1978; Hardy et al., 1983; Jahoda et al., 1984). Mesenchymal factors that are likely to play a role in this process include extracellular matrix components (Bissell et al., 1982), cell surfaceassociated proteins (Sariola et al., 1988; Hirai et al., 1992) and soluble growth factors such as KGF (Finch et al., 1989; Orr-Urtreger, 1991; Peters et al., 1992). Little is known about the mechanisms by which autocrine and paracrine growth factors act in concert to orchestrate the balance between growth and differentiation within a specialized cell type of a tissue. Recently, we utilized the human keratin 14 (K14) promoter to over-express an autocrine growth factor, TGFae, in the epidermis of transgenic mice (Vassar and Fuchs, 1991). At birth, TGFa mice were frail, had prematurely open eyelids and exhibited wrinkled skin that developed flakiness within several days. Epidermal hyperproliferation and thickening also occurred, and animals were smaller than normal (Vassar and Fuchs, 1991). Interestingly, these phenotypic differences disappeared at 5 weeks into postnatal development, concomitant with EGF receptor downregulation (Green et al., 1983; Vassar and Fuchs, 1991). In the present study, we focused on (i) exploring the role of KGF in mesenchymal -epithelial interactions and in keratinocyte growth and differentiation, and (ii) comparing the roles of paracrine and autocrine growth factors in epidermal growth and differentiation. To achieve these goals, we used the same human K14 promoter previously employed for our TGFoa studies, this time engineering transgenic mice that artificially expressed human KGF in their keratinocytes. This converted the action of KGF from paracrine to autocrine. Our results have uncovered some interesting and 973 -
L.Guo, Q.-C.Yu and E.Fuchs
K14 promoter ATG
hKGF 1
t Hinc 11
TM
i
hGH polyA+ /J1 ( 4 I1 E.IZA /Z )J-A Hind III 100 bp
Fig. 1. Transgene construct and K14-KGF transgenic mice. (Top) Human KGF (hKGF) cDNA was cloned into the BamHI site of an expression vector containing the human K14 promoter/enhancer (Vassar et al., 1989). Human growth hormone (hGH) sequences were also inserted into the XbaI site of this vector and used as intron-containing, 3' noncoding sequences and as a source of a polyadenylation signal. Abbreviations: ATG, translation start codon; TAA, termination codon; poly(A)+, polyadenylation signal. (A and A') Representative example of a newborn K14-KGF transgenic mouse (KGF-NB2) exhibiting a severe phenotype. Note grossly wrinkled skin and defined facial features relative to newborn control mouse in (B) and (B'). Note also that the transgenic mouse does not have whiskers (compare A' with B'). (C and C') A representative example of an -4 month old K14-KGF transgenic mouse (KGF-m86), relative to control littermate. Transgenic animals that reached adulthood were smaller than normal and exhibited fewer hairs around the eyelids, nose, limbs and abdomen. Note also that these animals had a wet undercoat, due to excessive salivary secretions.
major differences between KGF- and TGFat-mediated regulation of keratinocyte growth and differentiation, which included the appearance of gross transformations in tongue and epidermis of KGF-expressing animals as they aged. Perhaps even more importantly, our studies have yielded some completely unexpected and dramatic effects of keratinocyte KGF expression on hair follicle morphogenesis, adipogenesis and salivary gland differentiation, providing new and intriguing insights into the mechanisms underlying cell fate choices during development.
Results Newbom transgenic mice expressing keratin promoter-driven KGF exhibit grossly wrinkled skin and reduced hair follicle density The construct used in generation of our transgenic mice is outlined in Figure 1. It has previously been shown that bp of 5' upstream sequence of the human K14 gene was sufficient to drive expression of reporter genes in the basal cells of most stratified squamous epithelia, including epidermis (Vassar et al., 1989; Vassar and Fuchs, 1991). This promoter was selected to generate transgenic mice that could express human KGF cDNA in a keratinocyte-specific fashion. Human KGF cDNA was cloned from WI-38 human
~-2100-2300
974
fibroblast RNA using reverse transcription and polymerase chain reaction (PCR). The clone was sequenced in its entirety and corresponded exactly to the published sequence (Finch et al., 1989). The strategy of introducing human growth hormone (hGH) sequences as an intron-containing 3'-noncoding segment and polyadenylation signal was employed previously as a necessary measure to generate appreciable levels of transgene messages (Sandgren et al., 1990; Vassar and Fuchs, 1991; Cheng et al., 1992). The K14/KGF/hGH fusion gene was then used to generate transgenic mice as described previously (Vassar et al., 1989). Thirteen newborn mice from females carrying K14-KGF injected embryos were extremely frail, and either died or were sacrificed within 12-24 h of birth. These mice exhibited pronounced phenotypic aberrations (Figures 1A and lA', KGF NB-2t; compare with lB and 1B', control). Most notably, their skin was significantly wrinkled. In addition, the eyelids of transgenic animals were more pronounced than those of control littermates, although neither transgenic nor control eyelids were open at birth (Figure IA' and 1B'). Finally, the whiskers of wrinkled skin transgenic mice were often sparser than those of control animals, and in two cases, they were absent (Figure lA'; compare with tAbbreviations used in transgenic mice designations are: NB, newborn; m, male of > 12 days; f, female of > 12 days.
KGF and epithelial - mesenchymal interactions in vivo
.:f
c
A
t;
Co
-x
}> y
2uvB 3 o
'L
a
--).
cn
8
6
.1
.08
0
_
pg DNA
-nK
M_-JO,.;t
L.Guo, Q.-C.Yu and E.Fuchs
